This invention relates to a probes and methods of detecting and measuring enzyme-mediated breakdown of fibrinogen and fibrin. More particularly, the invention relates to a probe and a method for detecting degradation of fibrinogen and related substances mediated by fibrin(ogen)olytic matrix metalloproteinases.
The clotting of blood is part of the body's natural response to injury or trauma. Blood clot formation derives from a series of events called the coagulation cascade, in which the final steps involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen into fibrin, a mesh-like structure which forms the insoluble framework of the blood clot. As a part of hemostasis, clot formation is often a life-saving process in response to trauma and serves to arrest the flow of blood from severed vasculature.
The normally beneficial process of clot production in response to an injury can become life-threatening when it occurs at inappropriate places in the body. For example, a clot can obstruct a blood vessel and stop the supply of blood to an organ or other body part. In addition, the deposition of fibrin contributes to partial or complete stenosis of blood vessels, resulting in chronic diminution of blood flow. Equally life-threatening are clots that become detached from their original sites and flow through the circulatory system causing blockages at remote sites. Such clots are known as embolisms. Indeed, pathologies of blood coagulation, such as heart attacks, strokes, and the like, have been estimated to account for approximately fifty percent of all hospital deaths.
Fibrinogen is one of the more well-studied and abundant proteins in the human circulatory system. By the late 1960s, the general subunit structure of fibrinogen was firmly established (Blomback 1968) and, a decade later, the complete amino acid sequence was reported (Lottspeich et al. 1977; Henschen et al. 1977; Henschen et al. 1979; Doolittle et al. 1979). Over the next 10 years, the cluster of three separate genes encoding the .alpha. (alpha), .beta. (beta), and .gamma. (gamma) subunits was identified on chromosome 4q23-q32 (Kant et al. 1985), and the apparently complete genetic sequences of all three fibrinogen subunits were published (Chung et al. 1991).
Fibrinogen (also abbreviated herein as "Fg") is a heavily disulfide-bonded homodimeric protein, composed of two symmetrical units (monomers), each including single copies each or three polypeptide chains: the A.alpha. (alpha), B.beta. (beta), and .gamma. (gamma) chains. Thus, fibrinogen has the generic structure (A.alpha.B.beta..gamma.).sub.2. For a review see Doolittle (1987). All three of the fibrinogen subunits have coiled domains, which permit the subunits to engage one another to form a "coiled coil" region in the fibrinogen monomer. In addition, the B.beta. and .gamma. chains each have a globular domain, while the A.alpha. chain is present in two forms; a predominant form having no corresponding globular domain (A.alpha.), and a less prevalent form in which a globular domain is present (A.alpha..sub.E) (Fu et al. 1992; 1994). Accordingly, because fibrinogen is homodimeric and because two forms of the A.alpha. subunit have been identified, two principal forms of fibrinogen are recognized: (A.alpha.B.beta..gamma.).sub.2 and (A.alpha..sub.E B.beta..gamma.).sub.2.
Fibrinogen's complex structure, and its central role in blood clot formation and wound healing account for the high profile it has enjoyed as a subject of both biochemical and medical research. Recently, new attention has been given to structure/function relationships in the fibrinogen molecule. This new interest has in part been prompted by growth in the understanding of this protein's range of activity in normal and pathological states (Blomback 1991; Bini et al. 1992; Dvorak 1992). Moreover, antibodies have been developed which are specifically reactive with or specifically bind to only some of the fragments, thereby permitting molecular identification of certain fragments with great accuracy and precision (Kudryk et al. 1989a). However, despite these advances, the complexity of fibrinogen and its metabolic system have to date eluded complete elucidation.
Fibrinogen is synthesized and secreted into the circulation by the liver. Circulating fibrinogen is polymerized under attack by thrombin to form fibrin, which is the major component of blood clots or thrombi. Subsequently, fibrin is depolymerized under attack by plasmin to restore the fluidity of the plasma. Many of the steps in the polymerization and depolymerization processes have been well established (Doolittle 1984). The elevated levels of fibrinogen which are part of the acute phase response occurring in the wake of infections and trauma are now known to come from increased hepatic production, primarily in response to interleukin-6 (IL-6) (Sehgal et al. 1989).
In wound repair, fibrinogen serves as a key protein, achieving rapid arrest of bleeding following vessel injury. It promotes both the aggregation of activated platelets with one another to form a hemostatic plug, as well as endothelial cell binding at the site of injury to seal the margins of the wound. As the most abundant adhesive protein in the blood, fibrinogen attaches specifically to platelets, endothelial cells and neutrophils via different integrins (Hynes 1992). Five putative receptor recognition domains on human fibrinogen, distributed over its three subunits, have been identified by in vitro and in vivo analyses (Kloczewiak et al. 1984; Cheresh et al. 1989; Loike et al. 1991; Farrell et al. 1992; Gonda et al. 1982; Ribes et al. 1989).
Elevated levels of fibrinogen have been found in patients suffering from clinically overt coronary heart disease, stroke and peripheral vascular disease. Although the underlying mechanisms remain speculative, recent epidemiological studies leave little doubt that plasma fibrinogen levels are an independent cardiovascular risk factor possessing predictive power which is at least as high as that of other accepted risk factors such as smoking, hypertension, hyperlipoproteinemia or diabetes (Ernst 1990; Ernst et al. 1993). The structure of fibrin has been analyzed extensively in vitro (Doolittle 1984). Only recently, however, has attention been paid to the molecular structure of human thrombi and atherosclerotic plaques with respect to fibrinogen and fibrin products (Bini et al. 1987). Whereas thrombi formed in vivo consist primarily of fibrin II cross-linked by factor XIIIa, fibrinogen itself is a major component of uncomplicated atherosclerotic lesions, particularly fibrous and fatty plaques. Immunohistochemical as well as immunoelectrophoretic analyses indicate that fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue transglutaminase without becoming converted to fibrin (Valenzuela et al. 1992). Further understanding of these issues awaits the development of methods for the differential determination of fibrinogen subtypes in medical samples.
Fibrinogen-derived protein is also a major component of the stroma in which tumor cells are embedded, but little is known about its molecular structure. Tumor cells promote the secretion of potent permeability factors which cause leakage of fibrinogen from blood vessels (Dvorak et al. 1992). Extravascular clotting occurs due to procoagulants associated with tumor cells. The resulting fibrinogen/fibrin matrix is constantly remodeled during tumor growth as a consequence of fibrinolysis induced by tumor cell-derived plasminogen activators. It is assumed that fibrin/fibrinogen degradation products play a role during escape of metastatic tumor cells from the primary tumor. There are indications that integrin .alpha..sub.v .beta..sub.3, which is known to interact with the RGDS site in the C-terminal region of the .alpha. chain, may be an important tumor cell surface receptor since it is preferentially expressed on invasive melanoma (Felding-Habermann et al. 1992).
The formation of fibrin during inflammation, tissue repair, or hemostasis, plays only a temporary role and must be removed when normal tissue structure and function is restored. Thus, a fibrin clot that forms quickly to stop hemorrhage in an injured blood vessel is remodeled and then removed to restore normal blood flow as healing occurs. The system responsible for fibrin breakdown and clot removal is the fibrinolytic system. Action of the fibrinolytic system is tightly coordinated through the interaction of activators, zymogens, enzymes, as well as through inhibitors of each of these components, to provide focused local activation at sites of fibrin deposition (Francis et al. 1994; Collen 1980; Collen et al. 1991).
The principal mediator of fibrinolysis is plasmin, a trypsin-like endopeptidase which cleaves fibrin to dissolve clots and to permit injured tissues to regenerate. Plasmin has also been demonstrated to play a role in degrading proteins involved in cell-cell and cell-matrix interactions, as well as in activating other tissue remodeling enzymes such as matrix metalloproteinases (Murphy et al. 1992). In turn, control of plasmin activity, as well as these other extracellular events, is principally mediated by plasminogen activators, which convert the inactive zymogen plasminogen to the active enzyme plasmin.
Enzymes other than plasmin are also known which can degrade fibrin(ogen) to different extents. For example, endogenous leukocyte proteases (Bilezikian et al. 1977; Plow et al. 1975), later identified as elastase and cathepsin-G (Gramse et al. 1978; Plow 1980; Plow et al. 1982), can partially degrade fibrin(ogen). Exogenous enzymes are also known which degrade fibrin. Such enzymes include hemolytic enzymes collected from the venom of certain snakes, e.g., the families crotalidae and viperidae (Purves et al. 1987; Retzios et al. 1992; Sanchez et al. 1991). Fibrinolytic enzymes isolated from snakes can be grouped into two different classes (Guan et al. 1991). Those enzymes that preferentially degrade the A.alpha.-chain of fibrinogen and also the .alpha.- and .beta.-chains of fibrin are zinc metalloproteases (Guan et al. 1991) and all can be inhibited by EDTA. Enzymes in the second class are serine proteinases, and exhibit specificity for the .beta.-chain of fibrin (Guan et al. 1991). An endopeptidase from puff adder venom (Bitis arietans) can cleave at the .gamma.-chain cross-linking site and thereby cleave Fragment D-dimer into a D-like monomer (Purves et al. 1987). Fibrinolytic enzymes have also been obtained from leeches (Zavalova et al. 1993; Budzynski 1991), as well as from the growth medium of a bacterium (Aeromonas hydrophila) which was recovered from leech intestinal tract (Loewy et al. 1993).
Endogenous matrix metalloproteinases (MMPs) or "matrixins" include three classes of enzymes: collagenases, gelatinases, and stromelysins. MMPs are known to have the capacity to degrade a number of proteins and proteoglycans which are associated with the extracellular matrix (ECM) of connective tissue. They have been shown to break down a number of proteins including collagen (Types I-IV, VII and X), laminin, fibronectin, elastin and proteoglycans. MMPs have also been identified in leukocytes (Welgus et al. 1990). It has been shown that MMP-2 and MMP-9 possess elastase activity (Senior et al. 1991), to which some of the complex proteolytic activity, initially observed in granulocytes, could be attributed (Sterrenberg et al. 1983). MMPs participate in the remodeling of tissues in physiological processes such as morphogenesis and embryonic development, as well as in the pathophysiology of wound healing, tumor invasion, and arthritis (Matrisian 1992; Nagase et al. 1991; Woessner 1991; Werb et al. 1992).
From the foregoing discussion, it becomes clear that significant gaps exist in the understanding of processes involved in thrombus formation and degradation. While certain approaches have been identified which permit a measure of control over these processes, these approaches suffer serious deficiencies related to cost, efficacy, or safety. The diagnosis and treatment of disease states associated with physiological processes involving fibrinogen and fibrin have also been found lacking.
As a result, there exists a need for effective compositions and methods for use in illuminating the processes underlying thrombus development and thrombolysis, and for assessing these processes in vivo as they manifest as clot formation, embolism, atherosclerosis and the treatment of these processes
In addition, there exists a need for diagnostic and experimental materials and methods for revealing more information concerning the physical and chemical processes involved in thrombus formation and degradation.